Protein stability is now recognized as a central issue for the development and scale up of proteins, e.g, antibody molecules. The heavy and light chain variable domain sequences of antibodies are under selective pressure and can vary significantly in sequence from one antibody to another (Wu and Kabat, 1970). There are a large number of functional germline variable heavy (˜40; Tomlinson et al., 1992; Tomlinson et al., 1994; Matsuda and Honjo, 1996), variable kappa (˜40; Meindl et al., 1989; Cox et al., 1994; Barbie and Lefranc, 1998) and variable lambda (˜30; Williams et al., 1996; Kawasaki, et al., 1997) genes embedded within the human genome. The ability of the immune system to create many combinations of heavy chain and light chain variable domain germlines is one mechanism for creating antibody diversity (Edelman, 1959; Frañek, 1961). Additional diversity is derived by the insertion of a variable connecting peptide region, the J-connecting peptide (plus D-connecting peptide for heavy chains), between the variable domains and the constant domains (Leder, 1982). Subsequent to germline antibody selection against a specific antigen, B-cells expressing a single antibody with selected variable domain germlines and (D-)J-connecting peptides undergo the process of hypersomatic mutation within the variable heavy and light chain domains to increase antibody affinity towards the antigen (Leder, 1982; Tomlinson et al., 1996). Hypersomatic insertions or deletions within variable domains are also commonly observed, although at a much lower frequency than hypersomatic mutation (de Wildt et al., 1999). Thus, a mature antibody selected against a particular antigen will have highly unique variable domain sequences which are not necessarily optimized for stability.
Poor stability can affect the ability of an antibody or antibody domain to fold properly when expressed in various cellular systems, e.g., in bacterial or mammalian expression systems. Misfolding or poor stability can also result in fractional populations of non-functional material (Martsev et al., 1998) and/or antibodies with the tendency to form large soluble aggregates which are potentially dangerous or immunogenic when used therapeutically. Other stability problems include impeded refolding, degradation, impaired avidity, aggregation, or loss of activity following storage.
Stability problems are not limited to naturally occurring antibodies, but may occur in engineered antigen binding molecules as well, such as recombinant antibody libraries (Hoogenboom, 2005), antibody fragments (Holt et al., 2003; Todorovska et al., 2001; Worn and Plückthun, 2001; Reiter and Pastan, 1996) and engineered or humanized therapeutic antibodies for manufacturing and clinical purposes (Ewert et al., 2004; Carter and Merchant, 1997). In addition, multivalent forms of antibodies, such as bispecific molecules may have stability problems. While bispecific antibodies are desirable because of their therapeutic utility in humans, their production is unpredictable. For example, bispecific antibodies may result in substantial decrease in thermal stability, product yield, or in the formation of low molecular weight aggregates.
Unnatural changes to the VH and VL domains may also arise when humanizing rodent antibodies. Humanization is generally performed by grafting rodent complementarity determining regions (CDRs) onto the most similar human germline variable domain (Hurle and Gross, 1994). The germlines chosen for humanization may not be used with great frequency by the immune system and changes within the human frameworks are often necessary to achieve adequate antigen binding.
Unstable proteins suffer from many problems, including one or more of: unsuitability for scale-up production in bioreactors (e.g., because of low yield, significant levels of unwanted byproducts such as unassembled product, and/or aggregated material), difficulties in protein purification, and unsuitability for pharmaceutical preparation and use (e.g., owing to significant levels of breakdown product, poor product quality, and/or unfavorable pharmacokinetic properties). Instability within the variable domains of antibodies, Fabs, Fvs, or single-chain Fvs (scFvs) may result in many of these problems. scFv constructs in particular have demonstrated problems with stability, solubility, expression, aggregation, breakdown products, and overall manufacturability in both bacterial and in mammalian expression systems (see Worn and Pluckthun, 2001). Additionally, incorporation of scFv molecules into otherwise stable recombinant antibody products often imparts these generally undesirable traits to the new recombinant design.
Accordingly, there is a need for improved methods of predicting the stability of proteins, such as antibodies, and for improved, stable antigen binding molecules that are suitable for scalable production. There is also a need for improved methods that allow for scalable production of a population of stable antigen binding molecules that are suitable for pharmaceutical applications.